Dissociated cell cultures of Xenopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channels and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ, the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurites of cultured neurons were typical of young, embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport.